Tgfbeta-rII promoter polymorphisms

ABSTRACT

Disclosed are single nucleotide polymorphisms (SNPs) associated with end stage renal disease, breast cancer, lung cancer, and prostate cancer. Also disclosed are methods for using SNPs to determine susceptibility to these diseases; nucleotide sequences containing SNPs; kits for determining the presence of SNPs; and methods of treatment or prophylaxis based on the presence of SNPs.

BACKGROUND

This invention relates to detection of individuals at risk forpathological conditions based on the presence of single nucleotidepolymorphisms (SNPs).

During the course of evolution, spontaneous mutations appear in thegenomes of organisms. It has been estimated that variations in genomicDNA sequences are created continuously at a rate of about 100 new singlebase changes per individual (Kondrashow, J. Theor. Biol., 175:583-594,1995; Crow, Exp. Clin. Immunogenet., 12:121-128, 1995). These changes,in the progenitor nucleotide sequences, may confer an evolutionaryadvantage, in which case the frequency of the mutation will likelyincrease, an evolutionary disadvantage in which case the frequency ofthe mutation is likely to decrease, or the mutation will be neutral. Incertain cases, the mutation may be lethal in which case the mutation isnot passed on to the next generation and so is quickly eliminated fromthe population. In many cases, an equilibrium is established between theprogenitor and mutant sequences so that both are present in thepopulation. The presence of both forms of the sequence results ingenetic variation or polymorphism. Over time, a significant number ofmutations can accumulate within a population such that considerablepolymorphism can exist between individuals within the population.

Numerous types of polymorphisms are known to exist. Polymorphisms can becreated when DNA sequences are either inserted or deleted from thegenome, for example, by viral insertion. Another source of sequencevariation can be caused by the presence of repeated sequences in thegenome variously termed short tandem repeats (STR), variable numbertandem repeats (VNTR), short sequence repeats (SSR) or microsatellites.These repeats can be dinucleotide, trinucleotide, tetranucleotide orpentanucleotide repeats. Polymorphism results from variation in thenumber of repeated sequences found at a particular locus.

By far the most common source of variation in the genome are singlenucleotide polymorphisms or SNPs. SNPs account for approximately 90% ofhuman DNA polymorphism (Collins et al., Genome Res., 8:1229-1231, 1998).SNPs are single base pair positions in genomic DNA at which differentsequence alternatives (alleles) exist in a population. In addition, theleast frequent allele must occur at a frequency of 1% or greater.Several definitions of SNPs exist in the literature (Brooks, Gene,234:177-186, 1999). As used herein, the term “single nucleotidepolymorphism” or “SNP” includes all single base variants and so includesnucleotide insertions and deletions in addition to single nucleotidesubstitutions (e.g. A→G). Nucleotide substitutions are of two types. Atransition is the replacement of one purine by another purine or onepyrimidine by another pyrimidine. A transversion is the replacement of apurine for a pyrimidine or vice versa.

The typical frequency at which SNPs are observed is about 1 per 1000base pairs (Li and Sadler, Genetics, 129:513-523, 1991; Wang et al.,Science, 280:1077-1082, 1998; Harding et al., Am. J. Human Genet.,60:772-789, 1997; Taillon-Miller et al., Genome Res., 8:748-754, 1998).The frequency of SNPs varies with the type and location of the change.In base substitutions, two-thirds of the substitutions involve the C⇄T(G⇄A) type. This variation in frequency is thought to be related to5-methylcytosine deamination reactions that occur frequently,particularly at CpG dinucleotides. In regard to location, SNPs occur ata much higher frequency in non-coding regions than they do in codingregions.

SNPs can be associated with disease conditions in humans or animals. Theassociation can be direct, as in the case of genetic diseases where thealteration in the genetic code caused by the SNP directly results in thedisease condition. Examples of diseases in which single nucleotidepolymorphisms result in disease conditions are sickle cell anemia andcystic fibrosis. The association can also be indirect, where the SNPdoes not directly cause the disease but alters the physiologicalenvironment such that there is an increased likelihood that the patientwill develop the disease. SNPs can also be associated with diseaseconditions, but play no direct or indirect role in causing the disease.In this case, the SNP is located close to the defective gene, usuallywithin 5 centimorgans, such that there is a strong association betweenthe presence of the SNP and the disease state. Because of the highfrequency of SNPs within the genome, there is a greater probability thata SNP will be linked to a genetic locus of interest than other types ofgenetic markers.

Disease associated SNPs can occur in coding and non-coding regions ofthe genome. When located in a coding region, the presence of the SNP canresult in the production of a protein that is non-functional or hasdecreased function. More frequently, SNPs occur in non-coding regions.If the SNP occurs in a regulatory region, it may affect expression ofthe protein. For example, the presence of a SNP in a promoter region,may cause decreased expression of a protein. If the protein is involvedin protecting the body against development of a pathological condition,this decreased expression can make the individual more susceptible tothe condition.

Numerous methods exist for the detection of SNPs within a nucleotidesequence. A review of many of these methods can be found in Landegren etal., Genome Res., 8:769-776, 1998. SNPs can be detected by restrictionfragment length polymorphism (RFLP)(U.S. Pat. Nos. 5,324,631;5,645,995). RFLP analysis of the SNPs, however, is limited to caseswhere the SNP either creates or destroys a restriction enzyme cleavagesite. SNPs can also be detected by direct sequencing of the nucleotidesequence of interest. Numerous assays based on hybridization have alsobeen developed to detect SNPs. In addition, mismatch distinction bypolymerases and ligases has also been used to detect SNPs.

There is growing recognition that SNPs can provide a powerful tool forthe detection of individuals whose genetic make-up alters theirsusceptibility to certain diseases. There are four primary reasons whySNPs are especially suited for the identification of genotypes whichpredispose an individual to develop a disease condition. First, SNPs areby far the most prevalent type of polymorphism present in the genome andso are likely to be present in or near any locus of interest. Second,SNPs located in genes can be expected to directly affect proteinstructure or expression levels and so may serve not only as markers butas candidates for gene therapy treatments to cure or prevent a disease.Third, SNPs show greater genetic stability than repeated sequences andso are less likely to undergo changes which would complicate diagnosis.Fourth, the increasing efficiency of methods of detection of SNPs makethem especially suitable for high throughput typing systems necessary toscreen large populations.

SUMMARY

The present inventor has discovered novel single nucleotidepolymorphisms (SNPs) associated with the development of variousdiseases, including end stage renal disease, lung cancer, breast cancer,and prostate cancer. As such, these polymorphisms provide a method fordiagnosing a genetic predisposition for the development of thesediseases in individuals. Information obtained from the detection of SNPsassociated with the development of these diseases is of great value intheir treatment and prevention.

Accordingly, one aspect of the present invention provides a method fordiagnosing a genetic predisposition for end stage renal disease, lungcancer, breast cancer, or prostate cancer in a subject, comprisingobtaining a sample containing at least one polynucleotide from thesubject, and analyzing the polynucleotide to detect a geneticpolymorphism wherein said genetic polymorphism is associated with analtered susceptibility for end stage renal disease, lung cancer, breastcancer, or prostate cancer. In one embodiment, the polymorphism islocated in the TGF-β-RII gene.

Another aspect of the present invention provides an isolated nucleicacid sequence comprising at least 10 contiguous nucleotides from SEQ IDNO: 1, or their complements, wherein the sequence contains at least onepolymorphic site associated with a disease and in particular end stagerenal disease, lung cancer, breast cancer, or prostate cancer.

Yet another aspect of the invention is a kit for the detection of apolymorphism comprising, at a minimum, at least one polynucleotide of atleast 10 contiguous nucleotides of SEQ ID NO: 1, or their complements,wherein the polynucleotide contains at least one polymorphic siteassociated with end stage renal disease, lung cancer, breast cancer, orprostate cancer.

Yet another aspect of the invention provides a method for treating endstage renal disease, lung cancer, breast cancer, or prostate cancercomprising, obtaining a sample of biological material containing atleast one polynucleotide from the subject; analyzing the polynucleotideto detect the presence of at least one polymorphism associated with endstage renal disease, lung cancer, breast cancer, or prostate cancer; andtreating the subject in such a way as to counteract the effect of anysuch polymorphism detected.

Still another aspect of the invention provides a method for theprophylactic treatment of a subject with a genetic predisposition to endstage renal disease, lung cancer, breast cancer, or prostate cancercomprising, obtaining a sample of biological material containing atleast one polynucleotide from the subject; analyzing the polynucleotideto detect the presence of at least one polymorphism associated with endstage renal disease, lung cancer, breast cancer, or prostate cancer; andtreating the subject.

Further scope of the applicability of the present invention will becomeapparent from the detailed description and drawings provided below. Itshould be understood, however, that the following detailed descriptionand examples, while indicating preferred embodiments of the invention,are given by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from the following detaileddescription.

DEFINITIONS

-   nt=nucleotide-   bp=base pair-   kb=kilobase; 1000 base pairs-   ESRD=end-stage renal disease-   HTN=hypertension-   NIDDM=noninsulin-dependent diabetes mellitus-   CRF=chronic renal failure-   T-GF=tubulo-glomerular feedback-   CRG=compensatory renal growth-   MODY=maturity-onset diabetes of the young-   RFLP=restriction fragment length polymorphism-   MASDA=multiplexed allele-specific diagnostic assay-   MADGE=microtiter array diagonal gel electrophoresis-   OLA=oligonucleotide ligation assay-   DOL=dye-labeled oligonucleotide ligation assay-   SNP=single nucleotide polymorphism-   PCR=polymerase chain reaction

“polynucleotide” and “oligonucleotide” are used interchangeably and meana linear polymer of at least 2 nucleotides joined together byphosphodiester bonds and may consist of either ribonucleotides ordeoxyribonucleotides.

“sequence” means the linear order in which monomers occur in a polymer,for example, the order of amino acids in a polypeptide or the order ofnucleotides in a polynucleotide.

“polymorphism” refers to a set of genetic variants at a particulargenetic locus among individuals in a population.

“promoter” means a regulatory sequence of DNA that is involved in thebinding of RNA polymerase to initiate transcription of a gene. A “gene”is a segment of DNA involved in producing a peptide, polypeptide, orprotein, including the coding region, non-coding regions preceding(“leader”) and following (“trailer”) coding region, as well asintervening non-coding sequences (“introns”) between individual codingsegments (“exons”). A promoter is herein considered as a part of thecorresponding gene. Coding refers to the representation of amino acids,start and stop signals in a three base “triplet” code. Promoters areoften upstream (“5′ to”) the transcription initiation site of the gene.

“gene therapy” means the introduction of a functional gene or genes fromsome source by any suitable method into a living cell to correct for agenetic defect.

“wild type allele” means the most frequently encountered allele of agiven nucleotide sequence of an organism.

“genetic variant” or “variant” means a specific genetic variant which ispresent at a particular genetic locus in at least one individual in apopulation and that differs from the wild type.

As used herein the terms “patient” and “subject” are not limited tohuman beings, but are intended to include all vertebrate animals inaddition to human beings.

As used herein the terms “genetic predisposition”, “geneticsusceptibility” and “susceptibility” all refer to the likelihood that anindividual subject will develop a particular disease, condition ordisorder. For example, a subject with an increased susceptibility orpredisposition will be more likely than average to develop a disease,while a subject with a decreased predisposition will be less likely thanaverage to develop the disease. A genetic variant is associated with analtered susceptibility or predisposition if the allele frequency of thegenetic variant in a population or subpopulation with a disease,condition or disorder varies from its allele frequency in the populationwithout the disease, condition or disorder (control population) or acontrol sequence (wild type) by at least 1%, preferably by at least 2%,more preferably by at least 4% and more preferably still by at least 8%.

As used herein “isolated nucleic acid” means a species of the inventionthat is the predominate species present (i.e., on a molar basis it ismore abundant than any other individual species in the composition).Preferably, an isolated nucleic acid comprises at least about 50, 80 or90 percent (on a molar basis) of all macromolecular species present.Most preferably, the object species is purified to essential homogeneity(contaminant species cannot be detected in the composition byconventional detection methods).

As used herein, “allele frequency” means the frequency that a givenallele appears in a population.

Abbreviations used herein for nucleotides are the same as those in Table1 of MPEP section 2422 where a=adenine, g=guanine, c=cytosine,t=thyrnine, u=uracil, r=g or a, y=t/u or c, m=a or c, k=g or t/u, s=g orc, w=a or t/u, b=g or c or t/u, d=a or g or t/u, h=a or c or t/u, v=a org or c, and n=a or g or c or t/u, unknown, or other.

DETAILED DESCRIPTION

All publications, patents, patent applications and other referencescited in this application are herein incorporated by reference in theirentirety as if each individual publication, patent, patent applicationor other reference were specifically and individually indicated to beincorporated by reference.

TGF-β1 Signalling

Numerous animal and human studies have already linked the progression ofrenal disease, especially its hallmark pathology of interstitialfibrosis and glomerular sclerosis, to increased signalling by TGF-β1.Signalling by TGF-β1 involves specific binding of the ligand to the typeII TGF-β1 receptor (abbreviated as TGFβ-RII), present on the plasmamembrane of target cells such as fibroblasts in the case of glomerularand interstitial fibrosis. This receptor-ligand complex thenheterodimerizes with the type I TGF-β1 receptor (abbreviated asTGFβ-RI). TGFβ-RI is constitutively active. Like the concentrations ofligand (TGF-β1) and TGFβ-RI, the concentration of TGFβ-RII in the plasmamembrane ais likely to be rate-limiting for signalling by TGF-β1. Allelements of the pathway appear to be subject to complex regulation.

If the level of TGFβ-RII gene product (i.e., protein) is proportional tothe level of mRNA, and the mRNA level is proportional to thetranscriptional rate of the gene, then a SNP which disrupts atranscriptional activator site would be expected to decrease both therate of transcription of the gene and the eventual concentration ofTGFβ-RII in the plasma membrane of cells which express this protein. Thenet effect of such a SNP is expected to be protection against renalfailure.

TGF-β1 also inhibits cellular proliferation in a number of cell types.Signalling by TGF-β1 is thus expected to be depressed in individualswith a predisposition to malignancies.

Novel Polymorphisms

The present application provides four single nucleotide polymorphisms(SNPs) in genes associated with end stage renal disease due to NIDDM,lung cancer, breast cancer, or prostate cancer. All four polymorphismsare substitutions found on the TGF-β-RII promoter. The location of theseSNPs as well as the wild type and variant nucleotides is summarized inTable 7.

Preparation of Samples

The presence of genetic variants in the above genes or their controlregions, or in any other genes that may affect susceptibility to diseaseis determined by screening nucleic acid sequences from a population ofindividuals for such variants. The population is preferably comprised ofsome individuals with the disease, so that any genetic variants that arefound can be correlated with disease. The population is also preferablycomprised of some individuals that have known risk for the disease. Thepopulation should preferably be large enough to have a reasonable chanceof finding individuals with the sought-after genetic variant. As thesize of the population increases, the ability to find significantcorrelations between a particular genetic variant and susceptibility todisease also increases. Preferably, the population should have 10 ormore individuals.

The nucleic acid sequence can be DNA or RNA. For the assay of genomicDNA, virtually any biological sample containing genomic DNA (e.g. notpure red blood cells) can be used. For example, and without limitation,genomic DNA can be conveniently obtained from whole blood, semen,saliva, tears, urine, fecal material, sweat, buccal cells, skin or hair.For assays using cDNA or mRNA, the target nucleic acid must be obtainedfrom cells or tissues that express the target sequence. One preferredsource and quantity of DNA is 10 to 30 ml of anticoagulated whole blood,since enough DNA can be extracted from leukocytes in such a sample toperform many repetitions of the analysis contemplated herein.

Many of the methods described herein require the amplification of DNAfrom target samples. This can be accomplished by any method known in theart but preferably is by the polymerase chain reaction (PCR).Optimization of conditions for conducting PCR must be determined foreach reaction and can be accomplished without undue experimentation byone of ordinary skill in the art. In general, methods for conducting PCRcan be found in U.S. Pat. Nos. 4,965,188, 4,800,159, 4,683,202, and4,683,195; Ausbel et al., eds., Short Protocols in Molecular Biology,3^(rd) ed., Wiley, 1995; and Innis et al., eds., PCR Protocols, AcademicPress, 1990.

Other amplification methods include the ligase chain reaction (LCR)(see, Wu and Wallace, Genomics, 4:560-569, 1989; Landegren et al.,Science, 241:1077-1080, 1988), transcription amplification (Kwoh et al.,Proc. Natl. Acad Sci. USA, 86:1173-1177, 1989), self-sustained sequencereplication (Guatelli et al., Proc. Natl. Acad. Sci. USA, 87:1874-1878,1990), and nucleic acid based sequence amplification (NASBA). The lattertwo amplification methods involve isothermal reactions based onisothermal transcription, which produces both single stranded RNA(ssRNA) and double stranded DNA (dsDNA) as the amplification products ina ratio of about 30 or 100 to 1, respectively.

Detection of Polymorphisms

Detection of Unknown Polymorphisms

Two types of detection are contemplated within the present invention.The first type involves detection of unknown SNPs by comparingnucleotide target sequences from individuals in order to detect sites ofpolymorphism. If the most common sequence of the target nucleotidesequence is not known, it can be determined by analyzing individualhumans, animals or plants with the greatest diversity possible.Additionally the frequency of sequences found in subpopulationscharacterized by such factors as geography or gender can be determined.

The presence of genetic variants and in particular SNPs is determined byscreening the DNA and/or RNA of a population of individuals for suchvariants. If it is desired to detect variants associated with aparticular disease or pathology, the population is preferably comprisedof some individuals with the disease or pathology, so that any geneticvariants that are found can be correlated with the disease of interest.It is also preferable that the population be composed of individualswith known risk factors for the disease. The populations shouldpreferably be large enough to have a reasonable chance to findcorrelations between a particular genetic variant and susceptibility tothe disease of interest. In addition, the allele frequency of thegenetic variant in a population or subpopulation with the disease orpathology should vary from its allele frequency in the populationwithout the disease or pathology (control population) or the controlsequence (wild type) by at least 1%, preferably by at least 2%, morepreferably by at least 4% and more preferably still by at least 8%.

Determination of unknown genetic variants, and in particular SNPs,within a particular nucleotide sequence among a population may bedetermined by any method known in the art, for example and withoutlimitation, direct sequencing, restriction length fragment polymorphism(RFLP), single-strand conformational analysis (SSCA), denaturinggradient gel electrophoresis (DGGE), heteroduplex analysis (HET),chemical cleavage analysis (CCM) and ribonuclease cleavage.

Methods for direct sequencing of nucleotide sequences are well known tothose skilled in the art and can be found for example in Ausubel et al.,eds., Short Protocols in Molecular Biology, 3^(rd) ed., Wiley, 1995 andSambrook et al., Molecular Cloning, 2^(nd) ed., Chap. 13, Cold SpringHarbor Laboratory Press, 1989. Sequencing can be carried out by anysuitable method, for example, dideoxy sequencing (Sanger et al., Proc.Natl. Acad. Sci. USA, 74:5463-5467, 1977), chemical sequencing (Maxamand Gilbert, Proc. Natl. Acad. Sci. USA, 74:560-564, 1977) or variationsthereof. Direct sequencing has the advantage of determining variation inany base pair of a particular sequence.

RFLP analysis (see, e.g. U.S. Pat. Nos. 5,324,631 and 5,645,995) isuseful for detecting the presence of genetic variants at a locus in apopulation when the variants differ in the size of a probed restrictionfragment within the locus, such that the difference between the variantscan be visualized by electrophoresis. Such differences will occur when avariant creates or eliminates a restriction site within the probedfragment. RFLP analysis is also useful for detecting a large insertionor deletion within the probed fragment. Thus, RFLP analysis is usefulfor detecting, e.g., an Alu sequence insertion or deletion in a probedDNA segment.

Single-strand conformational polymorphisms (SSCPs) can be detected in<220 bp PCR amplicons with high sensitivity (Orita et al, Proc. Natl.Acad. Sci. USA, 86:2766-2770, 1989; Warren et al., In: Current Protocolsin Human Genetics, Dracopoli et al., eds, Wiley, 1994, 7.4.1-7.4.6.).Double strands are first heat-denatured. The single strands are thensubjected to polyacrylamide gel electrophoresis under non-denaturingconditions at constant temperature (i.e. low voltage and long run times)at two different temperatures, typically 4-10° C. and 23° C. (roomtemperature). At low temperatures (4-10° C.), the secondary structure ofshort single strands (degree of intrachain hairpin formation) issensitive to even single nucleotide changes, and can be detected as alarge change in electrophoretic mobility. The method is empirical, buthighly reproducible, suggesting the existence of a very limited numberof folding pathways for short DNA strands at the critical temperature.Polymorphisms appear as new banding patterns when the gel is stained.

Denaturing gradient gel electrophoresis (DGGE) can detect single basemutations based on differences in migration between homo- andheteroduplexes (Myers et al., Nature, 313:495-498, 1985). The DNA sampleto be tested is hybridized to a labeled wild type probe. The duplexesformed are then subjected to electrophoresis through a polyacrylamidegel that contains a gradient of DNA denaturant parallel to the directionof electrophoresis. Heteroduplexes formed due to single base variationsare detected on the basis of differences in migration between theheteroduplexes and the homoduplexes formed.

In heteroduplex analysis (HET) (Keen et al., Trends Genet.7:5, 1991),genomic DNA is amplified by the polymerase chain reaction followed by anadditional denaturing step which increases the chance of heteroduplexformation in heterozygous individuals. The PCR products are thenseparated on Hydrolink gels where the presence of the heteroduplex isobserved as an additional band.

Chemical cleavage analysis (CCM) is based on the chemical reactivity ofthymine (T) when mismatched with cytosine, guanine or thymine and thechemical reactivity of cytosine (C) when mismatched with thymine,adenine or cytosine (Cotton et al., Proc. Natl. Acad. Sci. USA,85:4397-4401, 1988). Duplex DNA formed by hybridization of a wild typeprobe with the DNA to be examined, is treated with osmium tetroxide forT and C mismatches and hydroxylamine for C mismatches. T and Cmismatched bases that have reacted with the hydroxylamine or osmiumtetroxide are then cleaved with piperidine. The cleavage products arethen analyzed by gel electrophoresis.

Ribonuclease cleavage involves enzymatic cleavage of RNA at a singlebase mismatch in an RNA:DNA hybrid (Myers et al., Science 230:1242-1246,1985). A ³²P labeled RNA probe complementary to the wild type DNA isannealed to the test DNA and then treated with ribonuclease A. If amismatch occurs, ribonuclease A will cleave the RNA probe and thelocation of the mismatch can then be determined by size analysis of thecleavage products following gel electrophoresis.

Detection of Known Polymorphisms

The second type of polymorphism detection involves determining whichform of a known polymorphism is present in individuals for diagnostic orepidemiological purposes. In addition to the already discussed methodsfor detection of polymorphisms, several methods have been developed todetect known SNPs. Many of these assays have been reviewed by Landegrenet al., Genome Res., 8:769-776, 1998 and will only be briefly reviewedhere.

One type of assay has been termed an array hybridization assay, anexample of which is the multiplexed allele-specific diagnostic assay(MASDA) (U.S. Pat. No. 5,834,181; Shuber et al., Hum. Molec. Genet.,6:337-347, 1997). In MASDA, samples from multiplex PCR are immobilizedon a solid support. A single hybridization is conducted with a pool oflabeled allele specific oligonucleotides (ASO). Any ASOs that hybridizeto the samples are removed from the pool of ASOs. The support is thenwashed to remove unhybridized ASOs remaining in the pool. Labeled ASOsremaining on the support are detected and eluted from the support. Theeluted ASOs are then sequenced to determine the mutation present.

Two assays depend on hybridization-based allele-discrimination duringPCR. The TaqMan assay (U.S. Pat. No. 5,962,233; Livak et al., NatureGenet., 9:341-342, 1995) uses allele specific (ASO) probes with a donordye on one end and an acceptor dye on the other end, such that the dyepair interact via fluorescence resonance energy transfer (FRET). Atarget sequence is amplified by PCR modified to include the addition ofthe labeled ASO probe. The PCR conditions are adjusted so that a singlenucleotide difference will effect binding of the probe. Due to the 5′nuclease activity of the Taq polymerase enzyme, a perfectlycomplementary probe is cleaved during the PCR while a probe with asingle mismatched base is not cleaved. Cleavage of the probe dissociatesthe donor dye from the quenching acceptor dye, greatly increasing thedonor fluorescence.

An alternative to the TaqMan assay is the molecular beacons assay (U.S.Pat. No. 5,925,517; Tyagi et al., Nature Biotech., 16:49-53, 1998). Inthe molecular beacons assay, the ASO probes contain complementarysequences flanking the target specific species so that a hairpinstructure is formed. The loop of the hairpin is complimentary to thetarget sequence while each arm of the hairpin contains either donor oracceptor dyes. When not hybridized to a donor sequence, the hairpinstructure brings the donor and acceptor dye close together therebyextinguishing the donor fluorescence. When hybridized to the specifictarget sequence, however, the donor and acceptor dyes are separated withan increase in fluorescence of up to 900 fold. Molecular beacons can beused in conjunction with amplification of the target sequence by PCR andprovide a method for real time detection of the presence of targetsequences or can be used after amplification.

High throughput screening for SNPs that affect restriction sites can beachieved by Microtiter Array Diagonal Gel Electrophoresis (MADGE) (Dayand Humphries, Anal. Biochem., 222:389-395, 1994). In this assayrestriction fragment digested PCR products are loaded onto stackablehorizontal gels with the wells arrayed in a microtiter format. Duringelectrophoresis, the electric field is applied at an angle relative tothe columns and rows of the wells allowing products from a large numberof reactions to be resolved.

Additional assays for SNPs depend on mismatch distinction by polymerasesand ligases. The polymerization step in PCR places high stringencyrequirements on correct base pairing of the 3′ end of the hybridizingprimers. This has allowed the use of PCR for the rapid detection ofsingle base changes in DNA by using specifically designedoligonucleotides in a method variously called PCR amplification ofspecific alleles (PASA) (Sommer et al., Mayo Clin. Proc., 64:1361-13721989; Sarker et al., Anal. Biochem. 1990), allele-specific amplification(ASA), allele-specific PCR, and amplification refractory mutation system(ARMS) (Newton et al., Nuc. Acids Res., 1989; Nichols et al., Genomics,1989; Wu et al., Proc. Natl. Acad. Sci. USA, 1989). In these methods, anoligonucleotide primer is designed that perfectly matches one allele butmismatches the other allele at or near the 3′ end. This results in thepreferential amplification of one allele over the other. By using threeprimers that produce two differently sized products, it can bedetermined whether an individual is homozygous or heterozygous for themutation (Dutton and Sommer, BioTechniques,11:700-702, 1991). In anothermethod, termed bi-PASA, four primers are used; two outer primers thatbind at different distances from the site of the SNP and two allelespecific inner primers (Liu et al., Genome Res., 7:389-398, 1997). Eachof the inner primers has a non-complementary 5′ end and form a mismatchnear the 3′ end if the proper allele is not present. Using this system,zygosity is determined based on the size and number of PCR productsproduced.

The joining by DNA ligases of two oligonucleotides hybridized to atarget DNA sequence is quite sensitive to mismatches close to theligation site, especially at the 3′ end. This sensitivity has beenutilized in the oligonucleotide ligation assay (Landegren et al.,Science, 241:1077-1080, 1988) and the ligase chain reaction (LCR;Barany, Proc. Natl. Acad. Sci. USA, 88:189-193, 1991). In OLA, thesequence surrounding the SNP is first amplified by PCR, whereas in LCR,genomic DNA can be used as a template.

In one method for mass screening for SNPs based on the OLA, amplifiedDNA templates are analyzed for their ability to serve as templates forligation reactions between labeled oligonucleotide probes (Samotiaki etal., Genomics, 20:238-242, 1994). In this assay, two allele-specificprobes labeled with either of two lanthanide labels (europium orterbium) compete for ligation to a third biotin labeled phosphorylatedoligonucleotide and the signals from the allele specificoligonucleotides are compared by time-resolved fluorescence. Afterligation, the oligonucleotides are collected on an avidin-coated 96-pincapture manifold. The collected oligonucleotides are then transferred tomicrotiter wells in which the europium and terbium ions are released.The fluorescence from the europium ions is determined for each well,followed by measurement of the terbium fluorescence.

In alternative gel-based OLA assays, numerous SNPs can be detectedsimultaneously using multiplex PCR and multiplex ligation (U.S. Pat. No.5,830,711; Day et al., Genomics, 29:152-162, 1995; Grossman et al., Nuc.Acids Res., 22:4527-4534, 1994). In these assays, allele specificoligonucleotides with different markers, for example, fluorescent dyes,are used. The ligation products are then analyzed together byelectrophoresis on an automatic DNA sequencer distinguishing markers bysize and alleles by fluorescence. In the assay by Grossman et al., 1994,mobility is further modified by the presence of a non-nucleotidemobility modifier on one of the oligonucleotides.

A further modification of the ligation assay has been termed thedye-labeled oligonucleotide ligation (DOL) assay (U.S. Pat. No.5,945,283; Chen et al., Genome Res., 8:549-556, 1998). DOL combines PCRand the oligonucleotide ligation reaction in a two-stage thermal cyclingsequence with fluorescence resonance energy transfer (FRET) detection.In the assay, labeled ligation oligonucleotides are designed to haveannealing temperatures lower than those of the amplification primers.After amplification, the temperature is lowered to a temperature wherethe ligation oligonucleotides can anneal and be ligated together. Thisassay requires the use of a thennostable ligase and a thermostable DNApolymerase without 5′ nuclease activity. Because FRET occurs only whenthe donor and acceptor dyes are in close proximity, ligation is inferredby the change in fluorescence.

In another method for the detection of SNPs termed minisequencing, thetarget-dependent addition by a polymerase of a specific nucleotideimmediately downstream (3′) to a single primer is used to determinewhich allele is present (U.S. Pat. No. 5,846,710). Using this method,several SNPs can be analyzed in parallel by separating locus specificprimers on the basis of size via electrophoresis and determining allelespecific incorporation using labeled nucleotides.

Determination of individual SNPs using solid phase minisequencing hasbeen described by Syvanen et al., Am. J Hum. Genet., 52:46-59, 1993. Inthis method the sequence including the polymorphic site is amplified byPCR using one amplification primer which is biotinylated on its 5′ end.The biotinylated PCR products are captured in streptavidin-coatedmicrotitration wells, the wells washed, and the captured PCR productsdenatured. A sequencing primer is then added whose 3′ end bindsimmediately prior to the polymorphic site, and the primer is elongatedby a DNA polymerase with one single labeled dNTP complementary to thenucleotide at the polymorphic site. After the elongation reaction, thesequencing primer is released and the presence of the labeled nucleotidedetected. Alternatively, dye labeled dideoxynucleoside triphosphates(ddNTPs) can be used in the elongation reaction (U.S. Pat. No.5,888,819; Shumaker et al., Human Mut., 7:346-354, 1996). In thismethod, incorporation of the ddNTP is determined using an automatic gelsequencer.

Minisequencing has also been adapted for use with microarrays (Shumakeret al., Human Mut., 7:346-354, 1996). In this case, elongation(extension) primers are attached to a solid support such as a glassslide. Methods for construction of oligonucleotide arrays are well knownto those of ordinary skill in the art and can be found, for example, inNature Genetics, Suppl., Vol. 21, January, 1999. PCR products arespotted on the array and allowed to anneal. The extension (elongation)reaction is carried out using a polymerase, a labeled DNTP andnoncompeting ddNTPs. Incorporation of the labeled dNTP is then detectedby the appropriate means. In a variation of this method suitable for usewith multiplex PCR, extension is accomplished with the use of theappropriate labeled ddNTP and unlabeled ddNTPs (Pastinen et al., GenomeRes., 7:606-614, 1997).

Solid phase minisequencing has also been used to detect multiplepolymorphic nucleotides from different templates in an undivided sample(Pastinen et al., Clin. Chem., 42:1391-1397, 1996). In this method,biotinylated PCR products are captured on the avidin-coated manifoldsupport and rendered single stranded by alkaline treatment. The manifoldis then placed serially in four reaction mixtures containing extensionprimers of varying lengths, a DNA polymerase and a labeled ddNTP, andthe extension reaction allowed to proceed. The manifolds are insertedinto the slots of a gel containing formamide which releases the extendedprimers from the template. The extended primers are then identified bysize and fluorescence on a sequencing instrument.

Fluorescence resonance energy transfer (FRET) has been used incombination with minisequencing to detect SNPs (U.S. Pat. No. 5,945,283;Chen et al., Proc. Natl. Acad. Sci. USA, 94:10756-10761, 1997). In thismethod, the extension primers are labeled with a fluorescent dye, forexample fluorescein. The ddNTPs used in primer extension are labeledwith an appropriate FRET dye. Incorporation of the ddNTPs is determinedby changes in fluorescence intensities.

The above discussion of methods for the detection of SNPs is exemplaryonly and is not intended to be exhaustive. Those of ordinary skill inthe art will be able to envision other methods for detection of SNPsthat are within the scope and spirit of the present invention.

In one embodiment the present invention provides a method for diagnosinga genetic predisposition for a disease. In this method, a biologicalsample is obtained from a subject. The subject can be a human being orany vertebrate animal. The biological sample must containpolynucleotides and preferably genomic DNA. Samples that do not containgenomic DNA, for example, pure samples of mammalian red blood cells, arehot suitable for use in the method. The form of the polynucleotide isnot critically important such that the use of DNA, cDNA, RNA or mRNA iscontemplated within the scope of the method. The polynucleotide is thenanalyzed to detect the presence of a genetic variant where such variantis associated with an increased risk of developing a disease, conditionor disorder, and in particular end stage renal disease, lung cancer,breast cancer, or prostate cancer. In one embodiment, the geneticvariant is located at one of the polymorphic sites contained in Table 7.In another embodiment, the genetic variant is one of the variantscontained in Table 7 or the complement of any of the variants containedin Table 7. Any method capable of detecting a genetic variant, includingany of the methods previously discussed, can be used. Suitable methodsinclude, but are not limited to, those methods based on sequencing, minisequencing, hybridization, restriction fragment analysis,oligonucleotide ligation, or allele specific PCR.

The present invention is also directed to an isolated nucleic acidsequence of at least 10 contiguous nucleotides from SEQ ID NO: 1, or thecomplements of SEQ ID NO 1. In one preferred embodiment, the sequencecontains at least one polymorphic site associated with a disease, and inparticular end stage renal disease, lung cancer, breast cancer, orprostate cancer. In one embodiment, the polymorphic site is selectedfrom the group contained in Table 7. In another embodiment, thepolymorphic site contains a genetic variant, and in particular, thegenetic variants contained in Table 7 or the complements of the variantsin Table 7. In yet another embodiment, the polymorphic site, which mayor may not also include a genetic variant, is located at the 3′ end ofthe polynucleotide. In still another embodiment, the polynucleotidefurther contains a detectable marker. Suitable markers include, but arenot limited to, radioactive labels, such as radionuclides, fluorophoresor fluorochromes, peptides, enzymes, antigens, antibodies, vitamins orsteroids.

The present invention also includes kits for the detection ofpolymorphisms associated with diseases, conditions or disorders, and inparticular end stage renal disease, lung cancer, breast cancer, orprostate cancer. The kits contain, at a minimum, at least onepolynucleotide of at least 10 contiguous nucleotides of SEQ ID NO 1, orthe complements of SEQ ID NO: 1. In one embodiment, the polynucleotidecontains at least one polymorphic site, preferably a polymorphic siteselected from the group contained in Table 7. Alternatively the 3′ endof the polynucleotide is immediately 5′ to a polymorphic site,preferably a polymorphic site contained in Table 7. In one embodiment,the polymorphic site contains a genetic variant, preferably a geneticvariant selected from the group contained in Table 7. In still anotherembodiment, the genetic variant is located at the 3′ end of thepolynucleotide. In yet another embodiment, the polynucleotide of the kitcontains a detectable label. Suitable labels include, but are notlimited to, radioactive labels, such as radionuclides, fluorophores orfluorochromes, peptides, enzymes, antigens, antibodies, vitamins orsteroids.

In addition, the kit may also contain additional materials for detectionof the polymorphisms. For example, and without limitation, the kits maycontain buffer solutions, enzymes, nucleotide triphosphates, and otherreagents and materials necessary for the detection of geneticpolymorphisms. Additionally, the kits may contain instructions forconducting analyses of samples for the presence of polymorphisms and forinterpreting the results obtained.

In yet another embodiment the present invention provides a method fordesigning a treatment regime for a patient having a disease, conditionor disorder and in particular end stage renal disease, lung cancer,breast cancer, or prostate cancer, caused either directly or indirectlyby the presence of one or more single nucleotide polymorphisms. In thismethod genetic material from a patient, for example, DNA, cDNA, RNA ormRNA is screened for the presence of one or more SNPs associated withthe disease of interest. Depending on the type and location of the SNP,a treatment regime is designed to counteract the effect of the SNP.

Alternatively, information gained from analyzing genetic material forthe presence of polymorphisms can be used to design treatment regimesinvolving gene therapy. For example, detection of a polymorphism thateither affects the expression of a gene or results in the production ofa mutant protein can be used to design an artificial gene to aid in theproduction of normal, wild type protein or help restore normal geneexpression. Methods for the construction of polynucleotide sequencesencoding proteins and their associated regulatory elements are well knowto those of ordinary skill in the art. Once designed, the gene can beplaced in the individual by any suitable means known in the art (GeneTherapy Technologies, Applications and Regulations, Meager, ed., Wiley,1999; Gene Therapy: Principles and Applications, Blankenstein, ed.,Birkhauser Verlag, 1999; Jain, Textbook of Gene Therapy, Hogrefe andHuber, 1998).

The present invention is also useful in designing prophylactic treatmentregimes for patients determined to have an increased susceptibility to adisease, condition or disorder, and in particular end stage renaldisease, lung cancer, breast cancer, or prostate cancer due to thepresence of one or more single nucleotide polymorphisms. In thisembodiment, genetic material, such as DNA, cDNA, RNA or mRNA, isobtained from a patient and screened for the presence of one or moreSNPs associated either directly or indirectly to a disease, condition,disorder or other pathological condition. Based on this information, atreatment regime can be designed to decrease the risk of the patientdeveloping the disease. Such treatment can include, but is not limitedto, surgery, the administration of pharmaceutical compounds ornutritional supplements, and behavioral changes such as improved diet,increased exercise, reduced alcohol intake, smoking cessation, etc.

EXAMPLES

Position of the single nucleotide polymorphism (SNP) is given accordingto the numbering scheme in GenBank Accession Number U37070. Thus, allnucleotides will be positively numbered, rather than bear negativenumbers reflecting their position upstream from the transcriptioninitiation site, a scheme often used for promoters. The two numberingsystems can be easily interconverted, if necessary. GenBank sequencescan be found at http://www.ncbi.nlm.nih.gov/

In the following examples, SNPs are written as “reference sequence” (or“wild type”) nucleotide”→“variant nucleotide.” Changes in nucleotidesequences are indicated in bold print. The standard nucleotideabbreviations are used in which A=adenine, C=cytosine, G=guanine,T=thymine, M=A or C, R=A or G. W=A or T, S=C or G, Y=C or T, K=G or T,V=A or C or G, H=A or C or T; D=A or G or T; B=C or G or T; N=A or C orG or T.

Example 1 Detection of Novel Polymorphisms by Direct Sequencing ofLeukocyte Genomic DNA

Leukocytes were obtained from human whole blood collected with EDTA asan anticoagulent. Blood was obtained from a group of black men, blackwomen, white men, and white women without any known disease. Blood wasalso obtained from individuals with end stage renal disease, lungcancer, breast cancer, or prostate cancer as indicated in the tablesbelow.

Genomic DNA was purified from the collected leukocytes using standardprotocols well known to those of ordinary skill in the art of molecularbiology (Ausubel et al., Short Protocol in Molecular Biology, 3^(rd)ed., John Wiley and Sons, 1995; Sambrook et al., Molecular Cloning, ColdSpring Harbor Laboratory Press, 1989; and Davis et al., Basic Methods inMolecular Biology, Elsevier Science Publishing, 1986). One hundrednanograms of purified genomic DNA was used in each PCR reaction.

Standard PCR reaction conditions were used. Methods for conducting PCRare well known in the art and can be found, for example, in U.S. Pat.Nos. 4,965,188, 4,800,159, 4,683,202, and 4,683,195; Ausbel et al.,eds., Short Protocols in Molecular Biology, 3^(rd) ed., Wiley, 1995; andInnis et al., eds., PCR Protocols, Academic Press, 1990. Specificprimers used are given in the following examples.

PCR reactions were carried out in a total volume of 50 ul containing10-15 ng leukocyte genomic DNA, 10 pmol of each primer, 200 nMdeoxynucleotide triphosphates (dNTPs), 1.25 U Taq polymerase (Qiagen),1× Qiagen PCR buffer (50 mM KCl, 10 mM Tris-HCl, pH 8.3, 1.5 mM MgCl₂,and 1×“Q” solution (Qiagen). After an initial 3 minutes denaturation at94° C., 35 cycles were performed consisting of 1 minute denaturation at94° C., 1 minute hybridization at 55° C., 2 minute extension at 72° C.,followed by a final extension step of 5 minutes at 72° C., and 1 minutecooling at 35° C.

Post-PCR clean-up was performed as follows. PCR reactions were cleanedto remove unwanted primer and other impurities such as salts, enzymes,and unincorporated nucleotides that could inhibit sequencing. One of thefollowing clean-up kits was used: Qiaquick-96 PCR Purification Kit(Qiagen) or Multiscreen-PCR Plates (Millipore, discussed below).

When using the Qiaquick protocol, PCR samples were added to the 96-wellQiaquick silica-gel membrane plate and a chaotropic salt, supplied as“PB Buffer,” was then added to each well. The PB Buffer causes DNA tobind to the membrane. The plate was put onto the Qiagen vacuum manifoldand vacuum was applied to the plate in order to pull sample and PBBuffer through the membrane. The filtrate was discarded. Next, thesamples were washed twice using “PE Buffer.” Vacuum pressure was appliedbetween each step to remove the buffer. Filtrate was similarly discardedafter each wash. After the last PE Buffer wash, maximum vacuum pressurewas applied to the membrane plate to generate maximum airflow throughthe membrane in order to evaporate residual ethanol left from the PEBuffer. The clean PCR product was then eluted from the filter using “EBBuffer.” The filtrate contained the cleaned PCR product and wascollected. All buffers were supplied as part of the Qiaquick-96 PCRPurification Kit. The vacuum manifold was also purchased from Qiagen forexclusive use with the Qiaquick-96 Purification Kit.

When using the Millipore Multiscreen-PCR Plates, PCR samples were loadedinto the wells of the Multiscreen-PCR Plate and the plate was thenplaced on a Millipore vacuum manifold. Vacuum pressure was applied for10 minutes, and the filtrate was discarded. The plate was then removedfrom the vacuum manifold and 100 μl of Milli-Q water was added to eachwell to rehydrate the DNA samples. After shaking on a plate shaker for 5minutes, the plate was replaced on the manifold and vacuum pressure wasapplied for 5 minutes. The filtrate was again discarded. The plate wasremoved and 60 μl Milli-Q water was added to each well to againrehydrate the DNA samples. After shaking on a plate shaker for 10minutes, the 60 μl of cleaned PCR product was transferred from theMultiscreen-PCR plate to another 96-well plate by pipetting. TheMillipore vacuum manifold was purchased from Millipore for exclusive usewith the Multiscreen-PCR plates.

Cycle sequencing was performed on the clean PCR product using an ABIPrism Big Dye Terminator Cycle Sequencing Ready Reaction kit(Perkin-Elmer). For a total volume of 20 μl, the following reagents wereadded to each well of a 96-well plate: 2.0 μl Terminator Ready Reactionmix, 3.0 μl 5× Sequencing Buffer (ABI), 5-10 μl template (30-90 ngdouble stranded DNA), 3.2 μM primer (primer used was the forward primerfrom the PCR reaction), and Milli-Q water to 20 μl total volume. Thereaction plate was placed into a Hybaid thermal cycler block andprogrammed as follows: ×1 cycle: 1 degree/sec thermal ramp to 94° C.,94° C. for 1 min; ×35 cycles: 1 degree/sec thermal ramp to 94° C., then94° C. for 10 sec, followed by 1 degree/sec thermal ramp to 50° C., then50° C. for 10 sec, followed by 1 degree/sec thermal ramp to 60° C., then60° C. for 4 minutes.

The cycle sequencing reaction product was cleaned up to remove theunincorporated dye-labeled terminators that can obscure data at thebeginning of the sequence. A precipitation protocol was used. To eachsequencing reaction in the 96-well plate 20 μl of Milli-Q water and 60μl of 100% isopropanol was added. The plate was left at room temperaturefor at least 20 minutes to precipitate the extension products. The platewas spun in a plate centrifuge (Jouan) at 3,000×g for 30 minutes.

Without disturbing the pellet, the supernatant was discarded byinverting the plate onto several paper tissues (Kimwipes) folded to thesize of the plate. The inverted plate, with Kimwipes in place, wasplaced into the centrifuge (Jouan) and spun at 700×g for 1 minute. TheKimwipes were discarded and the samples were loaded onto a sequencinggel.

Approximately 1 μl of sequencing product was loaded into each well of a96-lane 5% Long Ranger (FMC single pack) gel. The running bufferconsisted of 1×TBE. The glass plates consisted of ABI 48-cm plates foruse with a 96-lane 0.4 mm Mylar shark-tooth comb. A semi-automated ABIPrism 377-96 DNA sequencer was used (ABI 377 with 96-lane, Big Dyeupgrades). Sequencing run settings were as follows: run module 48E-1200,8 hr collection time, 2400 V electrophoresis voltage, 50 mAelectrophoresis current, 200 W electrophoresis power, CCD offset of 0,gel temperature of 51° C., 40 mW laser power, and CCD gain of 2.

The SEQUENCHER program (Gene Codes Corp., Ann Arbor, Mich.) was used toensure that only a high-quality sequence was used for allele assignment.The 5′ end of the sequence was trimmed to a maximum of 25%, until therewere fewer than 3 ambiguities. The 3′ end was defined as beginning 100bases after the trimmed 5′ end. The 3′ end was similarly trimmed toremove any sequence containing 3 or more ambiguities in 25 nucleotides.If any ambiguous bases still remained at the 5′ or 3′ end, they werealso removed. These settings are considerably stricter than the baselinedefault settings of the program. Individual sequences were excluded ifthey revealed less than 85% identity to the reference sequence (“dirtydata algorithm,” SEQUENCHER program).

Example 2 G to T Transversion at Position 945 of Human TGFβ-RII Promoter

TABLE 1 ALLELE FREQUENCIES G T CONTROL Black men (n = 22 chromosomes) 17(77%) 5 (23%) Black women (n = 28 chromosomes) 28 (100%) 0 (0%) Whitemen (n = 30 chromosomes) 28 (93%) 2 (7%) White women (n = 6 chromosomes) 4 (67%) 2 (33%) DISEASE BREAST CANCER Black women (n = 8 chromosomes) 8 (100%) 0 (0%) White women (n = 4 chromosomes)  4 (100%) 0 (0%) LUNGCANCER Black men (n = 12 chromosomes) 12 (100%) 0 (0%) Black women (n =14 chromosomes) 14 (100%) 0 (0%) White men (n = 6 chromosomes)  6 (100%)0 (0%) PROSTATE CANCER Black men (n = 6 chromosomes)  6 (100%) 0 (0%)White men (n = 12 chromosomes) 12 (100%) 0 (0%) ESRD due to NIDDM Blackmen (n = 6 chromosomes)  6 (100%) 0 (0%) Black women (n = 6 chromosomes) 6 (100%) 0 (0%) White men (n = 6 chromosomes)  6 (100%) 0 (0%) Whitewomen (n = 6 chromosomes)  6 (100%) 0 (0%)

TABLE 2 GENOTYPE FREQUENCIES G/G G/T T/T CONTROLS Black men (n = 11)  6(55%) 5 (45%) 0 (0%) Black women (n = 14) 14 (100%) 0 (0%) 0 (0%) Whitemen (n = 15) 13 (87%) 2 (13%) 0 (0%) White women (n = 3)  1 (33%) 2(67%) 0 (0%) DISEASE BREAST CANCER Black women (n = 4)  4 (100%) 0 (0%)0 (0%) White women (n = 2)  2 (100%) 0 (0%) 0 (0%) LUNG CANCER Black men(n = 6)  6 (100%) 0 (0%) 0 (0%) Black women (n = 7)  7 (100%) 0 (0%) 0(0%) White men (n = 3)  3 (100%) 0 (0%) 0 (0%) PROSTATE CANCER Black men(n = 3)  3 (100%) 0 (0%) 0 (0%) White men (n = 6)  6 (100%) 0 (0%) 0(0%) ESRD due to NIDDM Black men (n = 3)  3 (100%) 0 (0%) 0 (0%) Blackwomen (n = 3)  3 (100%) 0 (0%) 0 (0%) White men (n = 3)  3 (100%) 0 (0%)0 (0%) White women (n = 3)  3 (100%) 0 (0%) 0 (0%)

PCR and sequencing were conducted as in Example 1. The sense primer was5′-GGACATATCTGAAAGAGAAAGGGGG-3′ (SEQ ID NO: 2) and the antisense primerwas 5′- TTGGGAGTCACCTGAATGCTTG-3′ (SEQ ID NO: 3). The PCR productproduced spanned bases 892 to 1113 of the TGF-β-RII promoter.

As demonstrated above, the control samples all approximateHardy-Weinberg equilibrium. A frequency of 0.77 for the G allele (“p”)and 0.23 for the T allele (“q”) among black male control individualspredicts genotype frequencies of 59% G/G, 36% G/T, and 5% T/T atHardy-Weinberg equilibrium (p²+2pq+q²=1). The observed genotypefrequencies were 55% G/G, 45% G/T, and 0% T/T, in close agreement withthose predicted for Hardy-Weinberg equilibrium.

A frequency of 1.0 for the G allele (“p”) and 0 for the T allele (“q”)among black female control individuals predicts genotype frequencies of100% G/G, 0% G/T, and 0% T/T at Hardy-Weinberg equilibrium(p²+2pq+q²=1). The observed genotype frequencies were 100% G/G, 0% G/T,and 0% T/T, in perfect agreement with those predicted for Hardy-Weinbergequilibrium.

A frequency of 0.93 for the G allele (“p”) and 0.07 for the T allele(“q”) among white male control individuals predicts genotype frequenciesof 86% G/G, 14% G/T, and 0% T/T at Hardy-Weinberg equilibrium(p²+2pq+q²=1). The observed genotype frequencies were 87% G/G, 13% G/T,and 0% T/T, in very close agreement with those predicted forHardy-Weinberg equilibrium.

A frequency of 0.67 for the G allele (“p”) and 0.33 for the T allele(“q”) among white female control individuals predicts genotypefrequencies of 45% G/G, 44% G/T, and 11% T/T at Hardy-Weinbergequilibrium (p²+2pq+q²=1). The observed genotype frequencies were 33%G/G, 67% G/T, and 0% T/T, in fairly close agreement with those predictedfor Hardy-Weinberg equilibrium.

The frequency of the G allele, and especially of the G/G genotype, washigher than control frequencies for white women with breast cancer (Gallele frequency 100% vs. 67% control; G/G genotype frequency 100% vs.33% control), black men with lung cancer (G allele frequency 100% vs.77% control; G/G genotype frequency 100% vs. 55% control), white menwith lung cancer (G allele frequency 100% vs. 93% control; G/G genotypefrequency 100% vs. 87% control), black men with prostate cancer (Gallele frequency 100% vs. 77% control; G/G genotype frequency 100% vs.55% control), white men with prostate cancer (G allele frequency 100%vs. 93% control; G/G genotype frequency 100% vs. 87% control), black menwith ESRD due to NIDDM (G allele frequency 100% vs. 77% control; G/Ggenotype frequency 100% vs. 55% control), white men with ESRD due toNIDDM (G allele frequency 100% vs. 93% control; G/G genotype frequency100% vs. 87% control), and white women with ESRD due to NIDDM (G allelefrequency 100% vs. 67% control; G/G genotype frequency 100% vs. 33%control).

These data suggest that the reference allele (G) at this locuspredisposes white men and women, and black men to the followingdiseases: breast, lung, and prostate cancer, and ESRD due to NIDDM. Inother words, the SNP (T allele) is protective. Black women appear not tohave the T allele, so this locus appears to be neutral for them.However, from the data for the other three population groups (white andblack men, and white women), it is likely that the T allele predisposesblack women to breast and lung cancer, as well as ESRD due to NIDDM.

The G945→T SNP does not disrupt any known transcriptional regulatorysite. To be consistent with current models of increased TGFβ1 signallingas a cause of renal failure, and decreased TGFβ1 signalling as a causeof cancer, as yet unknown transcriptional repressor(s) and activator(s)are predicted to bind to this region of the TGFβ-RII promoter.

Example 3 G to M (A or C) Substitution at Position 983 of Human TGFβ-RIIPromoter

TABLE 3 ALLELE FREQUENCIES G A C CONTROL Black men (n = 22 chromosomes)18 (82%) 4 (18%) 0 (0%) Black women (n = 30 chromosomes) 29 (97%) 1 (3%)0 (0%) White men (n = 30 chromosomes) 30 (100%) 0 (0%) 0 (0%) Whitewomen (n = 6 chromosomes)  3 (50%) 1 (17%) 2 (33%) DISEASE BREAST CANCERBlack women (n = 8 chromosomes)  8 (100%) 0 (0%) 0 (0%) White women (n =4 chromosomes)  4 (100%) 0 (0%) 0 (0%) LUNG CANCER Black men (n = 12chromosomes) 12 (100%) 0 (0%) 0 (0%) Black women (n = 14 chromosomes) 14(100%) 0 (0%) 0 (0%) White men (n = 6 chromosomes)  4 (67%) 2 (33%) 0(0%) PROSTATE CANCER Black men (n = 6 chromosomes)  6 (100%) 0 (0%) 0(0%) White men (n = 12 chromosomes) 12 (100%) 0 (0%) 0 (0%) ESRD due toNIDDM Black men (n = 6 chromosomes)  4 (67%) 0 (0%) 2 (33%) Black women(n = 6 chromosomes)  6 (100%) 0 (0%) 0 (0%) White men (n = 6chromosomes)  6 (100%) 0 (0%) 0 (0%) White women (n = 6 chromosomes)  6(100%) 0 (0%) 0 (0%)

TABLE 4 GENOTYPE FREQUENCIES G/G G/A A/A C/C CONTROLS Black men (n = 11) 9 (82%) 0 (0%) 2 (18%) 0 (0%) Black women (n = 15) 14 (93%) 1 (7%) 0(0%) 0 (0%) White men (n = 15) 15 (100%) 0 (0%) 0 (0%) 0 (0%) Whitewomen (n = 3)  1 (33%) 1 (33%) 0 (0%) 1 (33%) DISEASE BREAST CANCERBlack women (n = 4)  4 (100%) 0 (0%) 0 (0%) 0 (0%) White women (n = 2) 2 (100%) 0 (0%) 0 (0%) 0 (0%) LUNG CANCER Black men (n = 6)  6 (100%) 0(0%) 0 (0%) 0 (0%) Black women (n = 7)  7 (100%) 0 (0%) 0 (0%) 0 (0%)White men (n = 3)  2 (67%) 0 (0%) 1 (33%) 0 (0%) PROSTATE CANCER Blackmen (n = 3)  3 (100%) 0 (0%) 0 (0%) 0 (0%) White men (n = 6)  6 (100%) 0(0%) 0 (0%) 0 (0%) ESRD due to NIDDM Black men (n = 3)  2 (67%) 0 (0%) 0(0%) 1 (33%) Black women (n = 3)  3 (100%) 0 (0%) 0 (0%) 0 (0%) Whitemen (n = 3)  3 (100%) 0 (0%) 0 (0%) 0 (0%) White women (n = 3)  3 (100%)0 (0%) 0 (0%) 0 (0%)

PCR and sequencing were conducted as in Example 1. The primers were thesame as in Example 2. Most SNPs are biallelic, but the G983→M SNP isunusual in being triallelic.

As shown above, the control samples approximate Hiardy-Weinbergequilibrium. A frequency of 0.82 for the G allele (“p”) and 0.18 for theA allele (“q”) among black male control individuals predicts genotypefrequencies of 67% G/G, 30% G/A, and 3% A/A at Hardy-Weinbergequilibrium (p²+2pq+q²=1). The observed genotype frequencies were 82%G/G, 0% G/A, and 18% A/A, in distant agreement with those predicted forHardy-Weinberg equilibrium.

A frequency of 0.97 for the G allele (“p”) and 0.03 for the A allele(“q”) among black female control individuals predicts genotypefrequencies of 94% G/G, 6% G/A, and 0% A/A at Hardy-Weinberg equilibrium(p²+2pq+q²=1). The observed genotype frequencies were 100% G/G, 0% G/A,and 0% A/A, in fairly close agreement with those predicted forHardy-Weinberg equilibrium.

A frequency of 1.0 for the G allele (“p”) and 0 for the A allele (“q”)among white male control individuals predicts genotype frequencies of100% G/G, 0% G/A, and 0% A/A at Hardy-Weinberg equilibrium(p²+2pq+q²=1).The observed genotype frequencies were 100% G/G, 0% G/A, and 0% A/A, inperfect agreement with those predicted for Hardy-Weinberg equilibrium.

A frequency of 0.50 for the G allele (“pi”), 0.17 for the A allele(“P2”), and 0.33 for the C allele (“p₃”) among white female controlindividuals predicts genotype frequencies of 25% G/G, 17% G/A, 3% A/A,11% C/C, 11% A/C, and 33% G/C at Hardy-Weinberg equilibrium. Thesefrequencies can be obtained by expanding the expression(p₁A₁+p₂A₂+p₃A₃)², where p₁+p₂+p₃=1 (Daniel L. Hartl, A Primer ofPopulation Genetics, 2nd ed., Sinauer Associates, Inc., 35, 1988). Inthis case, allele A₁=G, A₂=A, and A₃=C. The genotype frequencies of A₁A₁(here, G/G), A₁A₂ (here, G/A), A₂A₂ (here, A/A), A₁A₃ (here, G/C), A₂A₃(here, A/C), and A₃A₃ (here, C/C) are predicted to be p₁ ², 2p₁p₂, p₂ ²,2p₁p₃, 2p₂p₃, and p₃ ², respectively. The observed genotype frequencieswere 33% G/G, 33% G/A, 0% A/A, and 33% C/C, in rather distant agreementwith those predicted for Hardy-Weinberg equilibrium.

Assuming as a general rule that a difference in allele or genotypefrequency of at least 10% is clinically significant, the followingobservations can be made. The reference G allele at this locus isincreased in frequency relative to the control group, as is the G/Ggenotype, for white women with breast cancer, black men with lungcancer, black men with prostate cancer, and white women with ESRD due toNIDDM. These data suggest that the G allele predisposes individuals tothe above diseases for the above population groups. The G allele isdecreased in frequency relative to controls for white men with lungcancer and black men with ESRD due to NIDDM; in the last group, there isthe appearance of an otherwise unusual C allele.

This locus appears to be neutral in effect (i.e., possess unchangedallele and genotype frequencies, relative to control individuals) forblack women with breast cancer or lung cancer, white men with prostatecancer, and black women and white men with ESRD due to NEDDM.

The G983→M SNP is predicted to disrupt a potential binding site forRFX1_(—)02 (X-box binding regulatory factor or RFX1; an X-box consistsof DNA of the sequence 5′-GTNRCC (0-3N)RGYAAC-3′ (SEQ ID NO. 4), (whereN is any nucleotide, R is a purine [A or G], and Y is a pyrimidine[C orT]). The 3′ terminus of this binding site ends at nucleotide 972 on the(−) strand. The consensus RFX1_(—)02 binding site consists of thesequence complementary to 5′-NNGTTRCYNNNGYNACNN-3′ (SEQ ID NO.5). Boththe G983→A and G983→C forms of this triallelic SNP replace the indicatedG in the core recognition sequence. RFX1_(—)02 binding sites occursomewhat frequently, 0.95 matches per 1000 base pairs of random genomicsequence in vertebrates.

Transcriptional regulation by RFX1 can be either positive or negative.An example of transcriptional repression mediated by RFX1 occurs whenRFX1 binds to a methylated site near the transcription initiation siteof the collagen alpha2(I) gene (Sengupta P K et al., J. Biol. Chem.274(51):36649-36655, 1999). Conversely, RFX activates expression ofmajor histocompatibility complex (MHC) class II genes; absence of RFX5results in bare lymphocyte syndrome (Brickey W J, et al., J. Immunol.163(12):6622-6630, 1999).

Besides being triallelic, the G983→M SNP is additionally complex. Thereference allele, G, is increased in frequency in some diseases butdecreased in others.

The frequency of the G allele is increased in breast cancer in whitewomen, lung cancer in black men, and prostate cancer in black men.Without being bound by theory, if one assumes that cancer results frominappropriately low TGF-1 signalling, presumably due in part todecreased transcription of the TGF -RII gene, then it follows that RFXacts normally to repress transcription of the TGF -RII gene in thesediseases and subpopulations. Replacement of the G by another allele (Aor C) would result in less repression of the TGF -RII gene. Put anotherway, the presence of the reference G allele would result in increasedrepression of the TGF -RII gene and hence less signalling by TGF-1.

Where the frequency of the G allele is decreased relative to controls,as in white men with lung cancer, consistency with the theory thatdecreased signalling by TGF-1 underlies cancer would suggest that RFXacts as a transcriptional activator of the TGF RII gene, rather than asa repressor.

The converse is predicted for ESRD due to NIDDM, a condition assumed toresult from increased, rather than decreased, signalling by TGF-1. Blackmen with this disease, in whom the G allele frequency is decreased,suggest that RFX may act as a transcriptional repressor normally, by thesame arguments as above. White women with ESRD due to NIDDM, however, inwhom the frequency of the G allele is increased relative to that ofcontrol individuals, would predict that RFX normally acts as atranscriptional activator in this subpopulation.

Example 4 G to W(A or T) Substitution at Position 1009 of Human TGFβ-RIIPromoter

TABLE 5 ALLELE FREQUENCIES G A T CONTROL Black men (n = 20 chromosomes)10 (50%) 10 (50%) 0 (0%) Black women (n = 30 chromosomes)  9 (30%) 21(70%) 0 (0%) White men (n = 30 chromosomes) 24 (80%)  6 (20%) 0 (0%)White women (n = 6 chromosomes)  4 (67%)  2 (33%) 0 (0%) DISEASE BREASTCANCER Black women (n = 8 chromosomes)  3 (38%)  5 (63%) 0 (0%) Whitewomen (n = 4 chromosomes)  3 (75%)  1 (25%) 0 (0%) LUNG CANCER Black men(n = 12 chromosomes)  2 (17%) 10 (83%) 0 (0%) Black women (n = 14chromosomes)  2 (14%) 12 (86%) 0 (0%) White men (n = 6 chromosomes)  6(100%)  0 (0%) 0 (0%) PROSTATE CANCER Black men (n = 6 chromosomes)  1(17%)  5 (83%) 0 (0%) White men (n = 12 chromosomes) 10 (83%)  2 (17%) 0(0%) ESRD due to NIDDM Black men (n = 6 chromosomes)  0 (0%)  4 (67%) 2(33%) Black women (n = 6 chromosomes)  3 (50%)  3 (50%) 0 (0%) White men(n = 6 chromosomes)  4 (67%)  2 (33%) 0 (0%) White women (n = 6chromosomes)  4 (67%)  0 (0%) 2 (33%)

TABLE 6 GENOTYPE FREQUENCIES G/G G/A A/A T/T CONTROLS Black men (n = 10) 3 (30%) 4 (40%) 3 (30%) 0 (0%) Black women (n = 15)  2 (13%) 5 (33%) 8(53%) 0 (0%) White men (n = 15) 10 (67%) 4 (27%) 1 (7%) 0 (0%) Whitewomen (n = 3)  1 (33%) 2 (67%) 0 (0%) 0 (0%) DISEASE BREAST CANCER Blackwomen (n = 4)  1 (25%) 1 (25%) 2 (50%) 0 (0%) White women (n = 2)  1(50%) 1 (50%) 0 (0%) 0 (0%) LUNG CANCER Black men (n = 6)  0 (0%) 2(33%) 4 (67%) 0 (0%) Black women (n = 7)  0 (0%) 2 (29%) 5 (71%) 0 (0%)White men (n = 3)  3 (100%) 0 (0%) 0 (0%) 0 (0%) PROSTATE CANCER Blackmen (n = 3)  0 (0%) 1 (33%) 2 (67%) 0 (0%) White men (n = 6)  4 (67%) 2(33%) 0 (0%) 0 (0%) ESRD due to NTDDM Black men (n = 3)  0 (0%) 0 (0%) 2(67%) 1 (33%) Black women (n = 3)  1 (33%) 1 (33%) 1 (33%) 0 (0%) Whitemen (n = 3)  1 (33%) 2 (67%) 0 (0%) 0 (0%) White women (n = 3)  1 (33%)G/T = 2 (67%)

PCR and sequencing were conducted as in Example 1. The primers were thesame as in Example 2. Most SNPs are biallelic, but the G1009→W SNP isunusual in being triallelic.

As show above, the control samples approximate Hardy-Weinbergequilibrium. A frequency of 0.50 for the G allele (“p”) and 0.50 for theA allele (“q”) among black male control individuals predicts genotypefrequencies of 25% G/G, 50% G/A, and 25% A/A at Hardy-Weinbergequilibrium (p²+2pq+q²=1). The observed genotype frequencies were 30%G/G, 40% G/A, and 30% A/A, in close agreement with those predicted forHardy-Weinberg equilibrium.

A frequency of 0.30 for the G allele (“p”) and 0.70 for the A allele(“q”) among black female control individuals predicts genotypefrequencies of 9% G/G, 42% G/A, and 49% A/A at Hardy-Weinbergequilibrium (p²+2pq+q²=1). The observed genotype frequencies were 13%G/G, 33% G/A, and 53% A/A, in reasonably close agreement with thosepredicted for Hardy-Weinberg equilibrium.

A frequency of 0.80 for the G allele (“p”) and 0.20 for the A allele(“q”) among white male control individuals predicts genotype frequenciesof 64% G/G, 32% G/A, and 4% A/A at Hardy-Weinberg equilibrium(p²+2pq+q²=1). The observed genotype frequencies were 67% G/G, 27% G/A,and 7% A/A, in close agreement with those predicted for Hardy-Weinbergequilibrium.

A frequency of 0.67 for the G allele (“p”) and 0.33 for the A allele(“q”) among white female control individuals predicts genotypefrequencies of 45% G/G, 44% G/A, and 11% A/A at Hardy-Weinbergequilibrium (p²+2pq+q²=1). The observed genotype frequencies were 33%G/G, 67% G/A, and 0% A/A, in fair agreement with those predicted forHardy-Weinberg equilibrium.

Assuming as a general rule that a difference in allele or genotypefrequency of at least 10% is clinically significant, the followingobservations can be made. For black women with breast cancer, thefrequency of the G allele was increased relative to controls, suggestingthat the reference G allele contributes to breast cancer in black women.The frequency of the G/G genotype was increased and the G/A genotypedecreased relative to controls, and also relative to that expected forHardy-Weinberg equilibrium.

The G allele frequency for black women with breast cancer was 38%, vs.30% in controls. The expected genotype distribution according toHardy-Weinberg equilibrium was 9% G/G, 42% G/A, and 49% A/A for blackwomen. However, black women with breast cancer had a genotype frequencyof 25% G/G, almost three times higher than the 9% frequency expected,and twice the 13% observed in the control group. The frequency of theG/A genotype was only 25% among black women with breast cancer, ascompared to 42% predicted for Hardy-Weinberg equilibrium, and 33%observed in controls.

For white women with breast cancer, the G allele frequency was lessmarkedly increased than among black women: 75%, as compared to 67% incontrols. Conversely, the frequency of the A allele was slightlydecreased, from 33% in controls to 25% among white women with breastcancer. The expected genotype distribution according to Hardy-Weinbergequilibrium was 45% G/G, 44% G/A, and 11% A/A. The distribution ofgenotypes for white women with breast cancer was 50% G/G, 50% G/A, 0%A/A, again showing a slight excess of G/G and G/A genotypes at theexpense of the A/A genotype. These data suggest that the G allele alsopredisposes white women to breast cancer, although not to the samedegree as black women.

For white men with lung cancer, the situation is similar to breastcancer. White men with lung cancer have a marked increase in thefrequency of the reference G allele relative to controls, 100% vs. 80%.The distribution of genotypes for white men with lung cancer (100% G/G)in no way resembles the predicted Hardy-Weinberg distribution (64% G/G,32% G/A, 4% A/A), nor the observed distribution among controlindividuals (67% G/G, 27% G/A, 7% A/A). These data suggest that the Gallele strongly predisposes white men to lung cancer.

The story is different for African-Arnericans with lung cancer. Bothblack men and women have a markedly decreased frequency of the G allelerelative to control, 0% vs. 50% for black male controls and 30% forblack female controls. Conversely, the frequency of the A allele isincreased among black men and women with lung cancer. This can best beseen by looking at the frequency of the A/A genotype. It is 67% in blackmen with lung cancer, more than twice as much as the 25% predicted forblack men at Hardy-Weinberg equilibrium, and the 30% observed amongblack male controls. Similarly, the frequency of the A/A genotype is 71%among black women with lung cancer, as compared to only 49% predictedfor black women at Hardy-Weinberg equilibrium, and the 53% observedamong black female controls. These data suggest that the A allelestrongly predisposes black men and women to lung cancer.

For prostate cancer, the deviation from control allele frequencies ismuch more marked for black men than white men. The G allele frequency isdecreased nearly three-fold among black men with prostate cancer, 17%,as compared to 50% for control individuals. The frequency of the G/Ggenotype is reduced to 0% for black men with prostate cancer, ascompared to 25% predicted by Hardy-Weinberg equilibrium, and 30%observed among control individuals. These data suggest that the G alleleis protective against prostate cancer in black men, or alternatively,that the A allele predisposes to prostate cancer in black men. Thefrequency of the A/A genotype is 67% among black patients, over twicethe A/A frequency predicted for Hardy-Weinberg equilibrium (25%) as wellas that observed among control individuals (30%). For white men withprostate cancer, the allele and genotype frequencies are essentially thesame as control.

For black and white men with ESRD due to NIDDM, the frequency of the Gallele is markedly decreased relative to control, suggesting that the Gallele is protective against this disease in men. The G allele frequencyis 0% for black men with ESRD due to NIDDM, vs. 50% for controlindividuals. The A allele, on the other hand, has a frequency of 67%among black men with ESRD due to NIDDM, vs. 50% among controls. A secondSNP, the T allele at position 1009 in the TGF RII promoter, which doesnot occur at all in the control group, is present at a frequency of 33%among black men with ESRD due to NIDDM. The A and T alleles, therefore,appear to confer predisposition to ESRD due to NIDDM for black men.

White men with ESRD due to NIDDM similarly have over a two-fold lowerfrequency of the reference G allele compared to control individuals, 33%vs. 80%, suggesting that the G allele is protective against disease forwhite men. White men with ESRD due to NIDDM did not have the T allele;the A allele appears to be the major disease-predisposing allele forwhite men.

Black women with ESRD due to NIDDM have a higher frequency of the Gallele, 50% relative to control individuals whose G allele frequency isonly 30%. The G allele appears to strongly predispose black women toESRD due to NIDDM, in contrast to the protective effect of the G allelefor white and black men.

White women with ESRD due to NIDDM, like black men with the disease,have a 33% frequency of the T allele. The T allele does not appear atall among control individuals. Thus, the T allele strongly predisposewhite women to ESRD due to NIDDM.

The GI009→W SNP does not disrupt any known transcriptional regulatorysite. Control at this site is expected to be extremely complex,involving both activator(s) and repressor(s) of transcription, since thereference allele (G) can either contribute to, or protect against,disease depending on ethnicity (e.g. black vs. white men with lungcancer) or gender (e.g. black men vs. women with ESRD due to NIDDM).TABLE 7 Gene Region Location Wild Type Variant SEQ ID TGFβ-RII Promoter945 G T 1 983 G M 1 1009 G W 1Conclusion

In light of the detailed description of the invention and the examplespresented above, it can be appreciated that the several aspects of theinvention are achieved.

It is to be understood that the present invention has been described indetail by way of illustration and example in order to acquaint othersskilled in the art with the invention, its principles, and its practicalapplication. Particular formulations and processes of the presentinvention are not limited to the descriptions of the specificembodiments presented, but rather the descriptions and examples shouldbe viewed in terms of the claims that follow and their equivalents.While some of the examples and descriptions above include someconclusions about the way the invention may fuinction, the inventor doesnot intend to be bound by those conclusions and functions, but puts themforth only as possible explanations.

It is to be further understood that the specific embodiments of thepresent invention as set forth are not intended as being exhaustive orlimiting of the invention, and that many alternatives, modifications,and variations will be apparent to those of ordinary skill in the art inlight of the foregoing examples and detailed description. Accordingly,this invention is intended to embrace all such alternatives,modifications, and variations that fall within the spirit and scope ofthe following claims.

1. A method for diagnosing a genetic susceptibility for a disease,condition, or disorder in a subject comprising: obtaining a biologicalsample containing nucleic acid from said subject; and analyzing saidnucleic acid to detect the presence or absence of a single nucleotidepolymorphism in the TGFβ-RII gene, wherein said single nucleotidepolymorphism is associated with a genetic predisposition for a disease,condition or disorder selected from the group consisting of end stagerenal disease, lung cancer, breast cancer, and prostate cancer.
 2. Themethod of claim 1, wherein the gene TGFβ-RII comprises SEQ ID NO:
 1. 3.The method of claim 1, wherein said nucleic acid is DNA, RNA, cDNA ormRNA.
 4. The method of claim 2, wherein said single nucleotidepolymorphism is located at position 945, 983 or 1009 of SEQ ID NO:
 1. 5.The method of claim 4, wherein said single nucleotide polymorphism isselected from the group consisting of G945→T, G983→M, and G1009→W andthe complements thereof namely C945→A, C983→K, and C1009→W.
 5. Themethod of claim 1, wherein said analysis is accomplished by sequencing,mini sequencing, hybridization, restriction fragment analysis,oligonucleotide ligation assay or allele specific PCR.
 6. An isolatedpolynucleotide comprising at least 10 contiguous nucleotides of SEQ IDNO: 1, or the complement thereof, and containing at least one singlenucleotide polymorphism at position 945, 983, or 1009 of SEQ ID NO: 1wherein said at least one single nucleotide polymorphism is associatedwith a 5 disease, condition or disorder selected from the groupconsisting of end stage renal disease, lung cancer, breast cancer, andprostate cancer.
 7. The isolated polynucleotide of claim 7, wherein atleast one single nucleotide polymorphism is selected from the groupconsisting of G945→T, G983→M, and G1 009→W and the complements thereofnamely C945→A, C983→K, and C1009→W.
 8. The isolated polynucleotide ofclaim 7, wherein said at least one single nucleotide polymorphism islocated at the 3′ end of said nucleic acid sequence.
 9. The isolatedpolynucleotide of claim 7, further comprising a detectable label. 10.The isolated nucleic acid sequence of claim 10, wherein said detectablelabel is selected from the group consisting of radionuclides,fluorophores or fluorochromes, peptides, enzymes, antigens, antibodies,vitamins or steroids.
 11. A kit comprising at least one isolatedpolynucleotide of at least 10 contiguous nucleotides of SEQ ID NO: 1 orthe complement thereof, and containing at least one single nucleotidepolymorphism associated with a disease, condition, or disorder selectedfrom the group consisting of end stage renal disease, lung cancer,breast cancer, and prostate cancer; and instructions for using saidpolynucleotide for detecting the presence or absence of said at leastone single nucleotide polymorphism in said nucleic acid.
 12. The kit ofclaim 12 wherein said at least one single nucleotide polymorphism islocated at position 945, 983, or 1009 of SEQ ID NO:
 1. 13. The kit ofclaim 13 wherein said at least one single nucleotide polymorphism isselected from the group consisting of G945→T, G983→M, and G1009→W andthe complements thereof namely C945→A, C983→K, and C1009→W.
 14. The kitof claim 12, wherein said single nucleotide polymorphism is located atthe 3′ end of said polynucleotide.
 15. The kit of claim 12, wherein saidpolynucleotide further comprises at least one detectable label.
 16. Thekit of claim 16, wherein said label is chosen from the group consistingof radionuclides, fluorophores or fluorochromes, peptides enzymes,antigens, antibodies, vitamins or steroids.
 17. A kit comprising atleast one polynucleotide of at least 10 contiguous nucleotides of SEQ IDNO: 1 or the complement thereof, wherein the 3′ end of saidpolynucleotide is immediately 5′ to a single nucleotide polymorphismsite associated with a genetic predisposition to disease, condition, ordisorder selected from the group consisting of end stage renal disease,lung cancer, breast cancer, and prostate cancer; and instructions forusing said polynucleotide for detecting the presence or absence of saidsingle nucleotide polymorphism in a biological sample containing nucleicacid.
 18. The kit of claim 18, wherein said single nucleotidepolymorphism site is located at position 945, 983 or 1009 of SEQ IDNO:
 1. 19. The kit of claim 19, wherein said at least one polynucleotidefurther comprises a detectable label.
 20. The kit of claim 20, whereinsaid detectable label is chosen from the group consisting ofradionuclides, fluorophores or fluorochromes, peptides, enzymes,antigens, antibodies, vitamins or steroids.
 21. A method for treatmentor prophylaxis in a subject comprising: obtaining a sample of biologicalmaterial containing nucleic acid from a subject; analyzing said nucleicacid to detect the presence or absence of at least one single nucleotidepolymorphism in SEQ ID NO: 1 or the complement thereof associated with adisease, condition, or disorder selected from the group consisting ofend stage renal disease, lung cancer, breast cancer, and prostatecancer; and treating said subject for said disease, condition ordisorder.
 22. The method of claim 22 wherein said nucleic acid isselected from the group consisting of DNA, cDNA, RNA and mRNA.
 23. Themethod of claim 22, wherein said at least one single nucleotidepolymorphism is located at position 945, 983, or 1009 of SEQ ID NO: 1.24. The method of claim 22 wherein said at least one single nucleotidepolymorphism is selected from the group consisting of G945→T, G983→M,and G1009→W and the complements thereof namely C945→A, C983→K, andC1009→W.
 25. The method of claim 22 wherein said treatment counteractsthe effect of said at least one single nucleotide polymorphism detected.